Candida glabrata emerged in the 1990's as the second most important fungal pathogen, representing 10 to 30% of yeast isolates. Mortality rates up 53% have been reported for invasive C. glabrata infection, twice that observed with the most common pathogen, Candida albicans. C. glabrata is deficient in the virulence factors associated with C. albicans (dimorphism, adhesion, hydrolase secretion, and biofilm formation);rather, its high prevalence and mortality may be largely attributed to its intrinsic low-level resistance to widely used azole antifungals and its capacity for mutationally acquired high-level resistance. Studies from our lab and others revealed coordinately upregulated expression of azole/multidrug transporter genes CDR1 and PDH1 in most azole-resistant laboratory and clinical isolates, suggesting a common transcription factor. Indeed, sequence analysis of the single C. glabrata homolog of Saccharomyces cerevisiae Pdr1-Pdr3, "master regulators" of multidrug resistance, identified putative gain-of-function mutations in multiple azole-resistant isolates. C. glabrata PDR1 disruption, facilitated by a novel PCR-based method, and introduction of the mutated allele into a susceptible strain provided further support for its role in both intrinsic and acquired azole resistance. Moreover, microarray analysis has revealed Pdr1-mediated changes in gene expression beyond CDR1-PDH1 that may contribute to azole resistance and also alter C. glabrata virulence. Here we propose three Specific Aims that will characterize C. glabrata Pdr1 in depth. These include: (1) Pdr1 structure-function. Studies will include site-directed mutagenesis, protein domain interaction, the role of DNA repair in Pdr1 mutation, secondary structure analysis, and modeling Pdr1 evolution. (2) CDR1-PDH1 promoter structure-function. Regulatory and core elements within these promoters will be identified, and the Pdr1 response element (PDRE) characterized in terms of sequence, cooperativity between multiple elements, and evolutionary variation. (3) Regulators of Pdr1 activity. The roles in Pdr1 activation of MAP kinase Slt2, cAMP-dependent protein kinase A, histone acetyltransferease Gcn5, and related transcription factors such as Yrm1 will be evaluated. The proposed studies focused on "master regulator" Pdr1 will confer a deep understanding of the molecular basis for C. glabrata azole resistance, and provide a model for understanding similar resistance mechanisms in other pathogenic fungi. This is critical to the development of effective strategies for preventing or reversing resistance. PUBLIC HEALTH RELEVANCE Candida glabrata emerged in the 1990's as the second most important fungal pathogen, representing 10 to 30% of yeast clinical isolates, and with mortality rates up 53% for invasive infection. The high prevalence and mortality of C. glabrata infection may be largely attributed to its intrinsic low-level resistance to azoles, the most widely used group of antifungals, and its capacity for mutationally acquired high-level resistance. The proposed studies focus on C. glabrata Pdr1, "master regulator" of azole resistance;they will confer a deep understanding of the molecular basis for azole resistance, and could lead to novel strategies to prevent or reverse resistance.